[0001] This invention relates to purification and separation processes, for recovery and
isolation of biochemical macromolecular products from mixtures thereof and from media
in which they are contained or in which they have been produced. More particularly,
it relates to a process of biochemical purification which combines the processing
techniques of affinity chromatography and tangential flow ultrafiltration, and is
capable of being operated on a continuous-flow or semi-continuous-flow basis, for
use in the purification or separation of molecules of biological interest. The process
is particularly suited to the purification of hemoglobin and chemically-modified hemoglobin,
but is generally applicable to a wide range of biological and biochemical products.
[0002] Several processing techniques exist in the prior art to separate mixtures of molecules
of biological interest. One of the most powerful of these is affinity chromatography,
an extremely powerful separation method which separates on the basis of specific and
selective binding of molecules of interest to an affinity gel. Affinity gels typically
comprise a ligand-binding moiety immobilized on a gel support. Many types of well
characterized molecular interactions exist which can be exploited in affinity chromatography.
[0003] In GB-A-2178742 (Application No. 8615675, filed June 26, 1986) and various other
patent applications equivalent thereto and filed in other countries, there are described
methods for the purification of hemoglobin and its derivatives which have been chemically
modified to improve oxygen-carrying and circulatory characteristics. These methods
use affinity chromatography and are based upon the fact that native (oxy)hemoglobin
binds specifically to polyanionic moieties of certain affinity gels. Contrary to previous
beliefs, (oxy)hemoglobin as well as hemoglobin can be separated by affinity chromatography,
according to the aforementioned U.K. patent application. This is a particularly important
finding from a practical point of view, since it is virtually impossible to maintain
hemoglobin free from (oxy)hemoglobin in anything other than strictly controlled laboratory
conditions. This method is applicable to both positive and negative affinity absorption
mode. Positive affinity absorption mode is used to purify hemoglobin from various
sources, e.g. red blood cell lysates. When a mixture such as a red cell lysate is
passed through an affinity gel under conditions favouring hemoglobin binding to the
gel, hemoglobin is retained while the other components of the mixture are eluted.
Subsequent elution of the retained hemoglobin from the gel yields pure hemoglobin.
Negative affinity absorption mode is used to separate modified hemoglobin (i.e. hemoglobin
whose polyanion binding site has been chemically modified by the covalent attachment
of any of several known substances, for the purpose of ameliorating the undesirable
features of hemoglobin solutions as acellular oxygen-carrying fluids) from the residual
unmodified hemoglobin which remains due to the incomplete nature of the modification
reaction. In this process, unmodified hemoglobin is retained by the affinity gel while
modified hemoglobin, which cannot bind to the gel because its polyanion binding site
is by definition covalently occupied by the modifying agent, is eluted as the unretained
fraction. These methods both employ affinity chromatography columns which are highly
specific and, therefore, give very pure products. However, the affinity chromatography
is a relatively slow and laborious "batch" process.
[0004] Another known separation method is membrane ultrafiltration, which separates compounds
on the basis of molecular size. In the simplest form of this process, a solution is
poured through a filter with pores of a defined size, and those solutes which are
too large to pass through the pores are separated from those which can pass through.
This type of filtration is limited, however, by the fact that non-filterable solutes
accumulate on the filter and eventually block the flow of fluid through it.
[0005] This problem has recently been overcome in a new process known as tangential-flow
ultrafiltration (Gabler, F.R., ASM News, Vol, 50 No. 7 (1984), p. 299). In this process,
the solution flows parallel to the filter membrane so that the fluid flow continually
cleans the filter surface and prevents clogging by non-filterable solutes. A pressure
differential across the membrane causes fluid and filterable solutes to flow through
the filter. This can be conducted as a continuous-flow process, since the solution
is passed repeatedly over the membrance while that fluid which passes through the
filter is continually drawn off into a separate circuit. Since it separates solely
on the basis of molecular size, however, tangential-flow ultrafiltration lacks the
ability of selective separation based on biological specificity such as that used
in affinity chromatography.
[0006] The present invention involves use of tangential-flow filtration in a "continuous-flow"
separation process. In this process, separation of retained and unretained fractions
is achieved by differential filtration based upon molecular size. Affinity gel separation
is also employed, wherein an affinity gel is placed in the solution or mixture containing
the fractions to be separated, on the upstream side of the filter. Since the gel particles
are much larger than the solute particles, a filter can be selected which will allow
unbound solutes, i.e. solutes not bound to the gel, to pass through the filter, while
preventing the passage of gel particles and any substance bound to the gel.
[0007] According to the invention, there is provided a process for the separation of molecules
of biological interest from a mixture containing said molecules of interest, which
comprises subjecting said mixture to tangential flow ultrafiltration in the presence
of at least one macromolecular affinity substance which specifically binds said molecules
of interest and prevents their filtration while other smaller molecules are removed
by filtration.
[0008] Also according to the invention, there is provided a process of separating a first
biological macromolecule from a second biological macromolecule which comprises treating
a mixture containing both of said first and second biological macromolecules with
an affinity gel which selectively binds to one of said first and second macromolecules,
but not to the other thereof, and subsequently subjecting the mixture containing the
affinity gel bound macromolecules to tangential flow ultrafiltration, so as to obtain
affinity gel bound macromolecules in the retentate of said ultrafiltration and non-affinity
gel bound macromolecules in the filtrate of said ultrafiltration.
[0009] The term "biological macromolecule" used herein is intended to mean molecules of
at least about 1,000 daltons molecular weight, and of natural biological or biochemical
origin, or produced by biological or biochemical processes.
[0010] In the preferred form of the present invention, the processes of affinity gel separation
and tangential flow ultrafiltration are combined in a novel process called tangential
flow affinity ultrafiltration. In this combined process, a liquid solution or mixture
containing a component to be isolated is subjected to tangential flow ultrafiltration
in the presence of an appropriate affinity gel.
[0011] Affinity substances include soluble ligand-binding macromolecules and affinity gels.
For the purposes of purifying native and chemically modified hemoglobin, the basis
of the affinity property of the gel is the binding of native hemoglobin to the polyanionic
moiety of polyanion affinity gels, e.g. ATP-agarose. Isolation of the component of
interest is achieved by specifically binding it (a process conventionally known as
"positive" affinity chromatography) or by binding other components of the mixture
(conventionally known as "negative" affinity chromatography).
[0012] A wide variety of affinity gels suitable for use in the present invention are known,
and many are commercially available. The nature of the gel, the ligand to be attached
to the gel for purposes of binding to the selected biological macromolecule, and the
chemical manner of linking the ligand group to the gel must all be chosen with regard
to the nature of the selected biological macromolecule, the stability thereof towards
the reagents and solutions to which the affinity gel will be subjected in practice
of the invention, and the chemical removal of the biological macromolecules from the
affinity gel after the tangential flow ultrafiltration has taken place. Since most
processes of separation of biological macromolecules take place in aqueous medium,
stability of the gel and the ligand-gel linking groups and bonds towards water will
normally be important. The ligand group should be chosen to have a high degree of
specificity towards the biological macromolecule to be bound to the gel.
[0013] In the case where the biological macromolecule is hemoglobin or (oxy)hemoglobin,
the affinity gel preferably comprises a polyanionic-molecule linked by a spacer group
(cross-linking agent) to an affinity gel by known methods. Examples of polyanionic
ligands are diphosphoglycerate, nucleoside phosphates, inositol phosphates and sulphates,
etc. In fact, any polyanionic ligand, which is able to bind in the binding site or
cleft naturally occupied by DPG in hemoglobin, may be used. A wide variety or these
are known to those skilled in the art and published in the relevant scientific literature.
They include, in addition to those above, various organic phosphates, diphosphates
and polyphosphates, for example pyridoxal phosphate; phosphates of nucleic acids such
as ADP, ATP, guanosine phosphates, cytosine phosphates, thymine phosphates, uracil
phosphates, etc.; inositol phosphates; carbohydrate phosphates and sulfates; carbohydrate
mono- and poly-carboxylates, etc.
[0014] The ligand should not bind to the biological macromolecule so strongly that difficulties
of elution of the biological macromolecule from the gel are encountered. If the biological
macromolecule is too tightly bound, then denaturing reactions may occur in the use
of the necessarily strong reagents or conditions for elution, e.g. extreme pH. When
using organic polyphosphates as ligands to extract (oxy)hemoglobin, the bond strength
of the ligand to the (oxy)hemoglobin appears to increase with increasing numbers of
phosphate groups. The optimum bond strength appears to be derived from tri-, tetra-
and penta-phosphates, so that preferred ligand-forming compounds are nucleoside tri-
and tetra-phosphates, such as ATP, inositol tetraphosphate, inositol penta-phosphate
and the like, including mixtures thereof. The ligand molecule is chemically bonded
to the gel side groups in a manner such that the phosphate, sulphate or carboxylate
functional groups are left intact to react with the polyanion binding site in the
hemoglobin. In the case of ATP, it can be bound to the gel via its N-6 position or
8-position of adenosine, or through the periodate oxidized ribose moiety, for example.
[0015] The chromatographic gel is generally provided with a cross-linking agent or spacer,
which is effectively a chemical side chain group covalently linked to the gel at one
end and providing a reactive group on the other end for chemical attachment to the
ligand compound. While these spacers do not need to be linear chemical groups, they
should provide a spacing between the gel backbone and the reactive group for ligand
attachment of at least about 5 angstroms, and preferably at least 6 angstroms. In
practice, this means that they should contain a minimum of four linearly arranged
atoms separating the gel from the functional group. Otherwise, insufficient loading
of ligand groups onto the chromatographic gel might occur. The spacer groups should
be covalently linked, so that they are not broken during purification, regeneration
and sterilization processes. They should be stable and inert to all the components
of the mixture to be separated, and to the elution reagents to be used. Examples of
suitable reagents to be used to react with the gels to provide suitable spacer groups
are adipic acid, diaminohexane and derivatives thereof, such as adipic dihydrazide,
and various other, known diacids and diamino compounds.
[0016] The choice of suitable chromatographic gel is within the skill of the art, and can
be made from various commercially available products, provided that certain basic
criteria are observed. The gel needs to be substantially water insoluble, derivatizable
and non-toxic. It needs to be able to tolerate the regeneration conditions and processes
of treatment, for meeting sterile, sanitary administration for I.V. injection requirements.
Thus it must be readily sterilizable without impairment of its chemical properties.
Examples of suitable affinity gel supports are agarose and silica gels.
[0017] In general, modified cross-linked polysaccharide chromatographic gels are useful,
as exemplified by those commercially available from Pharmacia A.S. under the trade
names Sephadex, Sepharase, Sephacryl and Suparose. Commercially available chromatographic
silica gels and modified silica gels are also suitable. The gels in many cases are
available with spacer functional groups already attached. They can be obtained with
functional amino, epoxy or hydroxy groups. They may also, in addition, have an appropriate
ligand chemically attached to the spacer group, so that they are effectively ready
for use in the process of the present invention.
[0018] When the process of the invention is used to isolate biological macromolecules other
than hemoglobin and modified hemoglobins, a different selection of ligand may be made
to ensure its specificity towards the chosen biological macromolecule. Such selections
are generally within the skill of the art, based on relevant scientific literature
and the properties and characteristics of the specific biological macromolecule. Commonly,
the ligands will be biological substances themselves. For example, alpha-fetoprotein
from fetal sources is normally partially purified by antibody absorption (see, for
example, Nishi,
"Cancer Research"
30, October 1970, pp. 2507-2513; Ruoslahti, "
J.
Immunol"
121, 1687-1690 (1978); and Parmelee et al., "
J. Biol. Chemistry"
253 No. 7, April 10, 1978, pp. 2114-2119), but remains contaminated with albumin. The
removal of the albumin from alpha-fetoprotein can be accomplished by tangential flow
affinity ultrafiltration (TFAU) in the following manner. In this specific case, a
biological macromolecule with specific carbohydrate binding specificity, e.g. Concanavalin-A
(Con-A) can be linked to a suitable gel and used as affinity gel for the specific
separation of the glycoprotein alpha-fetoprotein from the non-glycoprotein albumin,
by TFAU in the presence of a Con-A agarose gel. Such a process can form part of the
procedure for isolating valuable alpha-fetoprotein from fetal serum and ambiotic fluid
(see, for example, Page,
Can. J. Biochem. 51, 1973, p. 1213). The process of TFAU according to the invention, using Con-A agarose
affinity gel, is generally applicable to the separation of glycoproteins from non-glycoproteins.
[0019] The filtration membrane for use in the tangential flow ultrafiltration is selected
so that the particles of the affinity substance, and, therefore, their complexes with
specifically bound components, are non-filterable, while the components of the mixture
to be separated are readily filterable. It is a feature of this process that two or
more simple or affinity gel ultrafiltration steps can be combined serially on a continuous-flow
basis to achieve complex separations.
[0020] The membranes useful in this process of tangential flow ultrafiltration in the present
invention are generally as described in the article by F. Raymond Gabler, previously
cited. They are generally synthetic membranes of either the microporous (MF) type,
the ultrafiltration (UF) type or the reverse osmosis (RO) type. An MF type has pore
sizes typically from 0.1 to 10 micrometers, and can be made so that it retains all
particles larger than the rated size. UF membranes have smaller pores and are characterized
by the size of the globular protein that will be retained. They are available in increments
from 1,000 to 1,000,000 nominal molecular weight (dalton) limits, corresponding approximately
to 0.001 to 0.05 micrometers. RO membranes are capable of retaining even smaller components,
and have effective port sizes in the range 0.0005 to 0.001 micrometers. For most applications
in separation of biological macromolecules, these port sizes are too small, and will
not allow passage even of non-gel-bound macromolecules. Ultrafiltration membranes
are most commonly suitable for use in the present invention. UF membranes are normally
asymmetrical with a thin film or skin on the upstream surface which is responsible
for their separating power. They are commonly made of synthetic polymer films, e.g.
polysulfone.
[0021] Membrane filters for tangential flow ultrafiltration are available as units, of different
configurations depending upon the volumes of liquid to be handled, and in a variety
of pore sizes. Particularly suitable for use in the present invention, on a relatively
large scale, are those known and commercially available as Millipore Prostack tangential
flow ultrafiltration units.
[0022] More specifically, according to a first process for separating/purifying native hemoglobin
from a hemoglobin-containing solution or mixture, for example, a red blood cell lysate,
the process of the invention is as follows. First, a simple tangential-flow filtration
step, using a membrane of approximately 110 kilo-dalton cut-off pore size, separates
the red cell stroma and large cytoplasmic structures from the cytoplasmic proteins
including hemoglobin. These proteins are then mixed with a polyanion affinity gel,
to which hemoglobin specifically binds, and then subjected to a second tangential
flow filtration step using the same membrane. Affinity gel-hemoglobin complexes are
prevented by their size from passing through the filter, while non-hemoglobin solutes
are filtered out.
[0023] In a third tangential flow filtration step, the gel-hemoglobin complexes are mixed
with a solution containing anionic or polyanionic molecules, such as ATP, phosphate
or sodium chloride, which compete with the polyanion originally bound to the gel for
binding to the hemoglobin. Hemoglobin is freed from the gel and passes through the
filter. This final filtration step yields pure hemoglobin. The gel can then be reused.
[0024] In a second process for separating/purifying modified hemoglobin from a reaction
mixture containing chemically modified hemoglobin as well as residual unmodified hemoglobin,
due to the incomplete nature of chemical modification reaction, the unmodified hemoglobin
must be removed before the solution can be considered for infusion, for example, ATP-hemoglobin
or as further modified for use as blood substitute as in the case of polymerization
of purified PLP-Hb. To achieve this, the reaction mixture is subjected to tangential
flow filtration in the presence of a polyanion affinity gel. Unmodified hemoglobin
whose polyanion binding site is unoccupied can bind to the gel and is, therefore,
prevented from passing through the filter due to its large molecular size. Modified
hemoglobin, whose polyanion binding site is by definition covalently occupied by the
modifying agent, cannot bind to the gel and is a small enough molecule to pass through
the filter. This yields pure modified hemoglobin which can be used immediately as
an acellular oxygen-carrier or subjected to further modification, e.g. polymerization
or cross-linking to various biopolymers. It is important that the incompleteness of
subsequent modification reactions is not essential, since the product purified by
the above process is free of native hemoglobin, which poses the greatest problems
in an intravascular solution. The unmodified hemoglobin can subsequently be recovered
from the gel by adding a competing anion as in the first process for recycling through
the modification process, and the gel can be reused.
[0025] The process of the present invention is well adapted for use on a commercial and
semi-commercial scale, as described in more detail below. It can be run semi-continuously,
i.e. on a continuous-flow basis of solution containing the desired biological macromolecule
bound to the affinity gel, past a tangential flow filter, until an entire, large batch
has thus been filtered, followed by a stage of continuous flow separation of gel from
desired biological macromolecule. Washing stages can be interposed between the filtration
stages. Then fresh batches of solution can be treated. In this way, a continuous,
cyclic process can be conducted, to give large yields of desired product, in acceptably
pure form, in relatively short periods of time. The unique features of affinity gel
with its ability to provide highly selective separation of biological macromolecules,
but only previously used in chromatographic mode, have been combined with the unique
features of tangential flow ultrafiltration with its ability to provide continuous
filtration of solids-containing solutions without filter clogging, to provide a unique
and highly advantageous process for the separation and purification of biological
macromolecular reaction products for use on a continuous basis and a commercial scale.
Moreover, the process is of very wide applicability. Whilst it shows great promise
is application to hemoglobin and hemoglobin derivatives, it is applicable to a wide
range of biological macromolecules, e.g. proteinaceous products of fermentation with
natural or genetically engineered microorganisms, high molecular weight antibiotics,
cellular secretions, etc.
[0026] In the accompanying drawings:
Figure 1A is a graph which illustrates the Tangential Flow Ultra Filtration (TAFU)
profile of stroma-free hemoglobin (SFH) in the presence of agarose-ATP (AGATP) gel,
derived from Example 1 below;
Figure 1B is a graph which illustrates the TAFU profile of a reaction mixture containing
pyridoxal phosphate modified SFH in the presence of AGATP gel, derived from Example
1 below;
Figures 2A and 2D are a series of graphs which characterize purified hemoglobin fractions
by High Performance Liquid Chromatography (HPLC), derived from Example 1 below; and
Figure 3 is a graph which illustrates the effects of TFAU on the oxygen binding affinity
of hemoglobin fractions purified according to the invention, and derived from Example
1 below;
Figure 4 is a diagrammatic process flow sheet of a commercial or semi-commercial facility
for putting the present invention into practice.
Example 1
[0027] Stroma-free hemoglobin (SFH) was purified using a tangential flow affinity ultrafiltration
(TFAU) technique.
[0028] SFH was prepared by a modified method of Rosenbery et al. (T.L. Rosenbery, J.F. Cheu,
M.M.L. Lee, T.A. Moulton and P. Onigman, J. Biochem. Biophys. Methods, 4, 39-48, 1981)
AGATP gels were prepared by a modified method of Lamed and Oplatka (R. Lamed and A.
Oplatka, Biochemistry, 13, 3137-3142, 1974).
[0029] The filtration apparatus used was an Amicon stir cell with a DIAFLO ultrafiltration
membrance, molecular weight cut-off 100 kilodaltons. This apparatus has a top valved
inlet for addition of liquid reagents, a top valved gas inlet for applying gas pressure,
a generally cylindrical body, an ultrafiltration membrane mounted generally horizontally
as the bottom wall of the body, and a fluid receiving chamber and radial liquid outlet
therefrom, below the membrane. There is provided a rotary stirrer within the body,
in the form of a flat paddle with planar, perpendicular blade faces just above the
level of the membrane, and magnetically operated from outside the cell. Thus, when
the stirrer rotates, it causes the liquid contents within the cell body to move continuously
across the surface of the membrane, in tangential flow mode. Positive pressure of
gas, normally nitrogen, applied through the top gas inlet, causes tangential flow
ultrafiltration to be effected through the membrane.
[0030] In this experiment, a filtration rate of 2 ml/minute, temperature 4°C and positive
nitrogen pressure of 50 psi were employed. Initially, the cell is charged with AGATP
and SFH, in a 50 mM Bis-Tris aqueous buffer, ph 7. The SFH binds to the gel to produce
a red gel. The filtrate issuing from the bottom of the cell is collected in successive,
separate fractions, and spectrometrically analysed for presence of the characteristic
hemoglobin red color (absorbance at 576 nm). The results are illustrated graphicaly
on Figure 1A. In section (a) of the graph, substantially all hemoglobin is retained
by the gel, early minor peaks of unbound hemoglobin-containing impurity being shown,
and by fraction fifteen, virtually none appears in the filtrate showing that it has
all been retained on the affinity gel or removed from the gel vicinity by tangential
flow ultrafiltration.
[0031] After collection of fifteen fractions, by which time the filtrate was colourless,
a competing affinity ligand (Buffer B) was added to the cell through the top liquid
inlet, e.g. 10 mM of ATP or 150 mM NaCl, as a buffer of 50 mM Bis-Tris pH 7.0. This
displaces the hemoglobin from the gel so that it instantly appears in pure form in
the filtrate from the cell by tangential flow ultrafiltration, as shown in section
(b) of Figure 1A.
Example 2
[0032] Chemically modified pyridoxal phosphate-modified hemoglobin (PLP-Hb) was separated
from unmodified stroma-free hemoglobin by tangential flow affinity ultrafiltration.
[0033] PLP-Hb was prepared by the method F. DeVenuto and A. Zegna (J. Surg. Res. 34, 205-212,
1983). Stroma-free hemoglobin and AGATP gels were prepared as previously indicated.
The apparatus and filtration conditions were as described in Example 1. The reaction
mixture of PLP-Hb (about 75% yield) containing SFH (or residual unmodified Hb) was
introduced into the cell and subjected to tangential flow ultrafiltration in the presence
of the AGATP. As illustrated in Figure 1B, the purification of this mixture gives
two major fractions, indicated as peaks
a and
b. PLP-Hb, the reaction product, with its diphosphoglycerate binding site occupied,
can no longer bind to the AGATP gel, so that it immediately passes through the membrane
by tangential flow affinity ultrafiltration, to give peak
a, whilst the unmodified Hb and SFH, i.e. the unreacted hemoglobin starting materials,
bind to the gel and do not pass through the filter. When all the PLP-Hb has passed
through the filter, as denoted by the colourless nature of the filtrate then being
obtained, Buffer B, as described in Example 1, is added to the cell. This effectively
detaches the SFH and unmodified hemoglobin from the gel, so that it now passes through
the filter, as denoted by peak
b. Note the similarity to the SFH peak in Figure 1A.
[0034] This example illustrates the use of the process of the present invention to effect
a clean separation of a modified hemoglobin product from unmodified hemoglobin, when
hemoglobin has been modified to form a blood substitute or an acellular oxygen-carrying
compound.
[0035] Figure 2 shows the high pressure liquid chromatography (HPLC) analysis of the SFH,
PLP-Hb reaction mixtures, AGATP retained and unretained fractions of the reaction
mixture. Figures 2A-2D are chromatograms of samples of (A) SFH (50 micrograms), (B)
PLP-Hb reaction mixture (100 micrograms), (C) retained fraction (50 micrograms), i.e.
peak
b from Figure 1B, and (D) unretained fraction (50 micrograms) (peak
a from Figure 1B) eluted from a HR 5/5 Mono S ion-exchange column (Pharmacia). In each
case, the solid line indicates the elution of the hemoglobin solutions by buffer A
(10 mM malonate, pH 5.7) followed by a linear gradient of buffer B (Buffer A plus
0.3 M lithium chloride, PH 5.7, broken line). Experimental conditions were: flow rate
0.5 ml/min, temperature 22°C, using a Pharmacia HPLC chromatographic system with Model
LCC-500 controller.
[0036] Pure SFH gives predominantly a single peak, as shown in Figure 2A. The SFH PLP-Hb
reaction mixture gives two clusters of peaks, as shown in Figure 2B. The second peak
occurring at high salt gradient compares with SFH in Figure 2A and the first peak(s)
eluting at lower concentrations of buffer B probably contain various species of PLP-Hb
(R. Benesch, R.E. Benesch and Suzanna Kwong, J. Biol. Chem., 275, 1320-1324, 1982).
Following TFAU of the reaction mixture as described above, the retained and unretained
fractions were similarly analysed by HPLC. Figure 2C relates to the retained fraction,
peak
b of Figure 1B, and is similar to SFH in Figure 2A and is predominantly SFH. The unretained
fraction, Figure 2D, is free of SFH and contains primarily PLP-Hb species. Note the
similarity of these peaks to the first peak(s) in Figure 2B. Thus the process TFAU
as described in these examples can produce highly purified PLP-Hb.
[0037] Figure 3 illustrates the oxygen dissociation curves of PLP-Hb reaction mixture (

.

.

) and TFAU-separated fractions a, PLP-Hb, (

), b, unmodified Hb, (- - - - -) and SFH (.....) in 50 mM Bis-Tris buffer at 37°C.
Curves were obtained using a Hem-O-Scan oxygen dissociation analyzer.
[0038] These oxygen dissociation curves indicate that the partial pressure of oxygen (P₅₀)
at which 50% of the hemoglobin is oxygenated is affected by the TFAU purification.
TFAU-purified PLP-Hb has a lower P₅₀(21mmHg) than the reaction mixture (15 mmHg).
The retained fraction (P₅₀ = 7 mmg) is similar to that of SFH (P₅₀ = 6 mmHg). Thus
TFAU effectively separates PLP-Hb from SFH in the reaction mixture, as characterized
by HPLC analysis and oxygen-releasing efficacy (i.e. lower P₅₀). In addition, removal
of unmodified SFH may have other beneficial effects, e.g. increased intravascular
half-life and reduced vasoconstrictive activity of the hemoglobin-based blood substitute.
[0039] It must be pointed out that any modified hemoglobin, such as ATP-Hb or glyoxylate-Hb,
can be purified by this technique. In addition, this method is a general purification
technology which may be applied to other biomacromolecules of interest.
[0040] Another aspect of this purification method is that it uses a 100 kilodalton cutoff
membrane filter which allows hemoglobin (diameter 5 nm) to pass through, it may be
capable of excluding infectious agents such as hepatitis B virus (42 nm diameter)
and the AIDS virus, HTLV-III (100 nm diameter). Thus the blood substitute prepared
from TFAU purified hemoglobin has the potential to be free of blood-borne viral disease.
[0041] With reference to Figure 4 of the accompanying drawings, this illustrates diagrammatically,
in flow sheet form, a semi-commercial or commercial facility for purification of a
biological product such as hemoglobin, according to the process of the present invention.
The process includes a first stage 40 in which red blood cells are washed, lysed and
filtered to remove cell residues and produce stroma-free hemoglobin (SFH). The process
also includes a second stage 50, operated successively to first stage 40, in which
the SFH is purified to obtain pure hemoglobin (Hb).
[0042] In the first stage 40, the starting material, red blood cells from which the hemoglobin
is to be extracted and purified, is stored in reservoir 41 in which it can be washed
and lysed with appropriate solution from vessel 42 in the known way. Then it is pumped
by means of pump 43 through outlet line 44 to the inlet side of a Millipore Prostack
tangential flow filter unit 45. The filtrate, namely SFH, is then fed from the outlet
side of filter unit 45 via line 46 to holding vessel 47, whilst the portion of the
material which does not pass through the filtration membrane of unit 45 (i.e. the
retentate) is recirculated via line 48 to reservoir 41 for recirculation through the
filter unit 45. Washing solution from vessel 42 can be used to wash reservoir 41 and
filter unit 45, after a batch of red blood cell ghosts have thus been washed and the
lysate moved onto holding vessel 47.
[0043] In the second stage 50, the combined process of affiity chromatography and tangential
flow ultrafiltration (TFAU) is employed, to produce pure hemoglobin. From the holding
vessel 47 of the first stage 40, a batch of the SFH is fed, in a first phase of operation,
via line 51 to a reaction vessel 52. There is provided a container 53 of washing solution
which can be fed via line 54 to reaction vessel 52 as and when desired, during washing
phases described below. The reaction vessel 52 contains an affinity gel, namely an
agarose-ATP affinity gel as described in GB-A-2178742.
[0044] In a first phase of operation of the process stage 50, this gel selectively binds
to the hemoglobin in reaction vessel 52. The liquid mixture containing the hemoglobin-gel
complex is then fed via outlet line 55 by means of pump 56 to the inlet side of a
Millipore Prostack tangential flow filter unit 57, essentially similar to that used
in the first stage 40. Here, the impurities including the modified hemoglobins and
the like which have not selectively bound to the gel are passed through the filter
as filtrate and fed out of the filter unit 57 via filtrate line 58 to receiving vessel
59, for discard or further utilization. Meanwhile, the Hb-gel complex does not pass
the filter and is returned as retentate via retentate line 60. Next, in a second constant
volume washing phase, the gel-Hb complex in reaction vessel 52 and filter unit 57
are washed with solution from container 53, the solution being chosen so that it does
not chemically dislodge the Hb from the gel in reaction vessel 52. The wash solution
passing the filter may be added to the filtrate in the first receiving vessel 59.
[0045] When the entire batch of SFH from reaction vessel 52 has thus been filtered and washed,
reaction vessel 52 contains gel-hemoglobin complexed perhaps mixed with some unreacted
gel, but substantially totally free of other impurities. Now it is required to remove
the Hb from the gel and recover the gel for reuse with another batch of SFH. Then,
in a third phase, salt solution, namely sodium chloride solution, is fed into reaction
vessel 52 from inlet port 61, located in by-pass line 62, and retentate line 60. In
reaction vessel 52, hemoglobin is replaced on the ATP gel by sodium ions, and the
hemoglobin in solution, along with the gel, passes via outlet line 55 to filter unit
57, through which it passes as filtrate and is led via filtrate line 58 to second
receiving vessel 63. The gel, meanwhile, exits the filter unit 57 via retentate line
60 to return to reaction vessel 52, where it is ready for re-use with another batch
of SFH from the first stage 40. A further constant volume washing cycle, using solution
from vessel 53, must be used before another batch of SFH is reacted to restore the
gel binding capacity, to minimize line contamination, filter contamination and the
like. Of course, appropriate valve controls are included, generally as illustrated,
to allow the process to be operated in the different phases and cycles with minimum
manual adjustments. The by-pass line 62 may equipped with appropriate controllers,
analytical instruments, sensors, samples, degassers and the like, to allow satisfactory
monitoring and control of the process as a whole.
[0046] Such a facility is readily capable of producing up to 2,400 units of hemoglobin per
8-hour run. It is, nevertheless, simple and inexpensive to construct and operate,
and capable of being built as a prefabricated or mobile facility, at a location of
immediate need for blood processing.
[0047] Moreover, the facility generally as illustrated is capable of extracting and purifying
a wide variety of biological molecules of interest, from solution in which they are
contained, in high or low concentration, such as fermentation solutions. In the first
stage 40, all debris, yeast residues, etc. from a fermentation process mixture can
be removed, to give a filtrate containing the desired product, but still in impure
form. This filtrate is then moved onto the second stage where the mixture contacts
an appropriately chosen affinity gel to which the desired product selectively binds.
Then the mixture is subjected to tangential flow affinity ultrafiltration (TFAU) to
remove the non-bound impurities, the gel is returned to the reaction vessel and the
desired product is removed from the gel, e.g. by salt elution and the mixture is again
subjected to tangential flow ultrafiltration to separate the desired product, in pure
form, from the gel.
[0048] By suitable adaptation of the operating stages and cycles, the facility as generally
illustrated is also capable of handling products prepared by so-called "negative affinity
chromatography", i.e. a process in which a desired biological substance is separated
from an undesired substance by use of an affinity gel which is selective to bind the
undesired substance. Then, the desired substance in the first phase of the second
stage is recovered as the filtrate from the tangential flow ultrafiltration unit 57,
in first receiving vessel 59. The undesired substance or impurity is subsequently
eluted from the gel and separated therefrom by the tangential flow affinity filtration
unit 57 in a second phase, and the gel thus prepared ready for re-use. Example 2 described
above, in which pyridoxal hemoglobin PLP-Hb is separated from unmodified hemoglobin
is a specific example of the use of the process of the invention in negative affinity
chromatography mode.
1. A process for the separation of molecules of biological interest from a mixture
containing said molecules of interest, which comprises subjecting said mixture to
tangential flow ultrafiltration in the presence of at least one macromolecular affinity
substance which specifically binds said molecules of interest and prevents their filtration
while other smaller molecules are removed by said filtration.
2. The process of claim 1 wherein said molecules of biological interest are biological
macromolecules.
3. The process of claim 2 wherein, subsequent to said tangential flow ultrafiltration
to remove smaller molecules by said filtration, said biological macromolecules are
displaced from binding with the macromolecular affinity substance, and the mixture
so formed is subjected to tangential flow ultrafiltration to separate the biological
macromolecules from the macromolecular affinity substance.
4. The process of claim 3 wherein said biological macromolecule of interest is hemoglobin
or a derivative thereof.
5. A process of separating a first biological macromolecule from a second biological
macromolecule which comprises treating a mixture containing both of said first and
second biological macromolecules with a macromolecular affinity substance which selectively
binds to one of said first and second macromolecules but not to the other thereof,
and subsequently subjecting the mixture containing the affinity substance-bound macromolecules
to tangential flow ultrafiltration, so as to obtain affinity substance-bound macromolecules
in the retentate of said ultrafiltration and non-affinity substance-bound macromolecules
in the filtrate of said ultrafiltration.
6. The process of claim 5, wherein, subsequent to the tangential flow ultrafiltration,
the affinity-substance-bound macromolecules in the retentate are chemically displaced
from binding with the macromolecular affinity substance, and the mixture of macromolecular
affinity substance and macromolecules so formed is subjected to tangential flow ultrafiltration
to separate the macromolecular affinity substance therefrom.
7. The process of claim 6, wherein the macromolecular affinity substance is subsequently
recycled to bind selectively to one of said first and second macromolecules from another
batch of mixture containing them.
8. The process of any of claims 5 to 7, wherein the macromolecular affinity substance
is an affinity gel.
9. The process of claim 8, wherein the affinity gel is an agarose gel or a silica
gel.
10. The process of any of claims 5 to 9, wherein the first and second biological macromolecules
are hemoglobin and hemoglobin derivatives.
11. The process of claim 10, wherein the affinity substance is an agarose gel having
a selective affinity binding anion attached thereto through a spacer chemical group
of at least 0.5 nm (5 Angstrom units).
12. The process of claim 11, wherein the selective affinity binding anion is an organic
phosphate, a nucleic acid phosphate, an inositol phosphate, a carbohydrate phosphate,
a carbohydrate sulfate, a carbohydrate monocarboxylate, a carbohydrate polycarboxylate
or diphosphoglycerate.
13. The process of any of claims 5 to 9, wherein the first and second biological macromolecules
are a glycoprotein and a non-glycoprotein.
14. The process of claim 13, wherein the affinity substance is agarose gel bound to
Concanavalin-A.